GABAB receptor-dependent modulation of network activity in the rat prefrontal cortex in vitro.

GABA (gamma-aminobutyric acid) can mediate inhibition via pre- and post/extrasynaptic GABA receptors. In this paper we demonstrate potentially post/extrasynaptic GABA(B) receptor-dependent tonic inhibition in L2/3 pyramidal cells of rat medial prefrontal cortex (mPFC) in vitro. First, we show via voltage-clamp experiments the presence of a tonic GABA(B) receptor-dependent outward current in these neurons. This GABA(B)ergic current could be induced by ambient GABA when present at sufficient concentrations. To increase ambient GABA levels in the usually silent slice preparation, we amplified network activity and hence synaptic GABA release with a modified artificial cerebrospinal fluid. The amplitude of tonic GABA(B) current was similar at different temperatures. In addition to the tonic GABA(B) current, we found presynaptic GABA(B) effects, GABA(B)-mediated inhibitory postsynaptic currents and tonic GABA(A) currents. Second, we performed current-clamp experiments to evaluate the functional impact of GABA(B) receptor-mediated inhibition in the mPFC. Activating or inactivating GABA(B) receptors led to rightward (reduction of excitability) or leftward (increase of excitability) shifts, respectively, of the input-output function of mPFC L2/3 pyramidal cells without effects on the slope. Finally, we showed in electrophysiological recordings and epifluorescence Ca(2+)-imaging that GABA(B) receptor-mediated tonic inhibition is capable of regulating network activity. Blocking GABA(B) receptors increased the frequency of excitatory postsynaptic currents impinging on a neuron and prolonged network upstates. These results show that ambient GABA via GABA(B) receptors is powerful enough to modulate neuronal excitability and the activity of neural networks.

Dopamine increases the gain of the input-output response of rat prefrontal pyramidal neurons.

Dopaminergic modulation of prefrontal cortical activity is known to affect cognitive functions like working memory. Little consensus on the role of dopamine modulation has been achieved, however, in part because quantities directly relating to the neuronal substrate of working memory are difficult to measure. Here we show that dopamine increases the gain of the frequency-current relationship of layer 5 pyramidal neurons in vitro in response to noisy input currents. The gain increase could be attributed to a reduction of the slow afterhyperpolarization by dopamine. Dopamine also increases neuronal excitability by shifting the input-output functions to lower inputs. The modulation of these response properties is mainly mediated by D1 receptors. Integrate-and-fire neurons were fitted to the experimentally recorded input-output functions and recurrently connected in a model network. The gain increase induced by dopamine application facilitated and stabilized persistent activity in this network. The results support the hypothesis that catecholamines increase the neuronal gain and suggest that dopamine improves working memory via gain modulation.

The dynamical response properties of neocortical neurons to temporally modulated noisy inputs in vitro.

Cortical neurons are often classified by current-frequency relationship. Such a static description is inadequate to interpret neuronal responses to time-varying stimuli. Theoretical studies suggested that single-cell dynamical response properties are necessary to interpret ensemble responses to fast input transients. Further, it was shown that input-noise linearizes and boosts the response bandwidth, and that the interplay between the barrage of noisy synaptic currents and the spike-initiation mechanisms determine the dynamical properties of the firing rate. To test these model predictions, we estimated the linear response properties of layer 5 pyramidal cells by injecting a superposition of a small-amplitude sinusoidal wave and a background noise. We characterized the evoked firing probability across many stimulation trials and a range of oscillation frequencies (1-1000 Hz), quantifying response amplitude and phase-shift while changing noise statistics. We found that neurons track unexpectedly fast transients, as their response amplitude has no attenuation up to 200 Hz. This cut-off frequency is higher than the limits set by passive membrane properties (approximately 50 Hz) and average firing rate (approximately 20 Hz) and is not affected by the rate of change of the input. Finally, above 200 Hz, the response amplitude decays as a power-law with an exponent that is independent of voltage fluctuations induced by the background noise.

Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats.

Calcium influx into the dendritic tufts of layer 5 neocortical pyramidal neurons modifies a number of important cellular mechanisms. It can trigger local synaptic plasticity and switch the firing properties from regular to burst firing. Due to methodological limitations, our knowledge about Ca2+ spikes in the dendritic tuft stems mostly from in vitro experiments. However, it has been speculated that regenerative Ca2+ events in the distal dendrites correlate with distinct behavioral states. Therefore it would be most desirable to be able to record these Ca2+ events in vivo, preferably in the behaving animal. Here, we present a novel approach for recording Ca2+ signals in the dendrites of populations of layer 5 pyramidal neurons in vivo, which ensures that all recorded fluorescence changes are due to intracellular Ca2+ signals in the apical dendrites. The method has two main features: 1) bolus loading of layer 5 with a membrane-permeant Ca2+ dye resulting in specific loading of pyramidal cell dendrites in the upper layers and 2) a fiberoptic cable attached to a gradient index lens and a prism reflecting light horizontally at 90 degrees to the angle of the apical dendrites. We demonstrate that the in vivo signal-to-noise ratio recorded with this relatively inexpensive and easy-to-implement fiberoptic-based device is comparable to conventional camera-based imaging systems used in vitro. In addition, the device is flexible and lightweight and can be used for recording Ca2+ signals in the distal dendritic tuft of freely behaving animals.

Combined voltage and calcium epifluorescence imaging in vitro and in vivo reveals subthreshold and suprathreshold dynamics of mouse barrel cortex.

Cortical dynamics can be imaged at high spatiotemporal resolution with voltage-sensitive dyes (VSDs) and calcium-sensitive dyes (CaSDs). We combined these two imaging techniques using epifluorescence optics together with whole cell recordings to measure the spatiotemporal dynamics of activity in the mouse somatosensory barrel cortex in vitro and in the supragranular layers in vivo. The two optical signals reported distinct aspects of cortical function. VSD fluorescence varied linearly with membrane potential and was dominated by subthreshold postsynaptic potentials, whereas the CaSD signal predominantly reflected local action potential firing. Combining VSDs and CaSDs allowed us to monitor the synaptic drive and the spiking activity of a given area at the same time in the same preparation. The spatial extent of the two dye signals was different, with VSD signals spreading further than CaSD signals, reflecting broad subthreshold and narrow suprathreshold receptive fields. Importantly, the signals from the dyes were differentially affected by pharmacological manipulations, stimulation strength, and depth of isoflurane anesthesia. Combined VSD and CaSD measurements can therefore be used to specify the temporal and spatial relationships between subthreshold and suprathreshold activity of the neocortex.

The impact of input fluctuations on the frequency-current relationships of layer 5 pyramidal neurons in the rat medial prefrontal cortex.

The role of irregular cortical firing in neuronal computation is still debated, and it is unclear how signals carried by fluctuating synaptic potentials are decoded by downstream neurons. We examined in vitro frequency versus current (f-I) relationships of layer 5 (L5) pyramidal cells of the rat medial prefrontal cortex (mPFC) using fluctuating stimuli. Studies in the somatosensory cortex show that L5 neurons become insensitive to input fluctuations as input mean increases and that their f-I response becomes linear. In contrast, our results show that mPFC L5 pyramidal neurons retain an increased sensitivity to input fluctuations, whereas their sensitivity to the input mean diminishes to near zero. This implies that the discharge properties of L5 mPFC neurons are well suited to encode input fluctuations rather than input mean in their firing rates, with important consequences for information processing and stability of persistent activity at the network level.

Extending lifetime of plastic changes in the human brain.

The ability of the brain to adjust to changing environments and to recover from damage rests on its remarkable capacity to adapt through plastic changes of underlying neural networks. We show here with an eye movement paradigm that a lifetime of plastic changes can be extended to several hours by repeated applications of theta burst transcranial magnetic stimulation to the frontal eye field of the human cortex. The results suggest that repeated application of the same stimulation protocol consolidates short-lived plasticity into long-lasting changes.

Repetitive TMS over the human oculomotor cortex: comparison of 1-Hz and theta burst stimulation.

The aim of the study was to compare the effect duration of two different protocols of repetitive transcranial magnetic stimulation (rTMS) on saccade triggering. In four experiments, two regions (right frontal eye field (FEF) and vertex) were stimulated using a 1-Hz and a theta burst protocol (three 30Hz pulses repeated at intervals of 100ms). The same number of TMS pulses (600 pulses) was applied with stimulation strength of 80% of the resting motor threshold for hand muscles. Following stimulation the subjects repeatedly performed an oculomotor task using a modified overlap paradigm, and saccade latencies were measured over a period of 60min. The results show that both 1-Hz and theta burst stimulation had inhibitory effects on saccade triggering when applied over the FEF, but not over the vertex. One-hertz rTMS significantly increased saccade latencies over a period of about 8min. After theta burst rTMS, this effect lasted up to 30min. Furthermore, the decay of rTMS effects was protocol-specific: After 1-Hz stimulation, saccade latencies returned to a baseline level much faster than after theta burst stimulation. We speculate that these time course differences represent distinct physiological mechanisms of how TMS interacts with brain function.

Predicting spike timing of neocortical pyramidal neurons by simple threshold models.

Neurons generate spikes reliably with millisecond precision if driven by a fluctuating current--is it then possible to predict the spike timing knowing the input? We determined parameters of an adapting threshold model using data recorded in vitro from 24 layer 5 pyramidal neurons from rat somatosensory cortex, stimulated intracellularly by a fluctuating current simulating synaptic bombardment in vivo. The model generates output spikes whenever the membrane voltage (a filtered version of the input current) reaches a dynamic threshold. We find that for input currents with large fluctuation amplitude, up to 75% of the spike times can be predicted with a precision of +/-2 ms. Some of the intrinsic neuronal unreliability can be accounted for by a noisy threshold mechanism. Our results suggest that, under random current injection into the soma, (i) neuronal behavior in the subthreshold regime can be well approximated by a simple linear filter; and (ii) most of the nonlinearities are captured by a simple threshold process.

Neocortical pyramidal cells respond as integrate-and-fire neurons to in vivo-like input currents.

In the intact brain neurons are constantly exposed to intense synaptic activity. This heavy barrage of excitatory and inhibitory inputs was recreated in vitro by injecting a noisy current, generated as an Ornstein-Uhlenbeck process, into the soma of rat neocortical pyramidal cells. The response to such in vivo-like currents was studied systematically by analyzing the time development of the instantaneous spike frequency, and when possible, the stationary mean spike frequency as a function of both the mean and the variance of the input current. All cells responded with an in vivo-like action potential activity with stationary statistics that could be sustained throughout long stimulation intervals (tens of seconds), provided the frequencies were not too high. The temporal evolution of the response revealed the presence of mechanisms of fast and slow spike frequency adaptation, and a medium duration mechanism of facilitation. For strong input currents, the slow adaptation mechanism made the spike frequency response nonstationary. The minimal frequencies that caused strong slow adaptation (a decrease in the spike rate by more than 1 Hz/s), were in the range 30-80 Hz and depended on the pipette solution used. The stationary response function has been fitted by two simple models of integrate-and-fire neurons endowed with a frequency-dependent modification of the input current. This accounts for all the fast and slow mechanisms of adaptation and facilitation that determine the stationary response, and proved necessary to fit the model to the experimental data. The coefficient of variability of the interspike interval was also in part captured by the model neurons, by tuning the parameters of the model to match the mean spike frequencies only. We conclude that the integrate-and-fire model with spike-frequency-dependent adaptation/facilitation is an adequate model reduction of cortical cells when the mean spike-frequency response to in vivo-like currents with stationary statistics is considered.

An Organotypic Spinal Cord - Dorsal Root Ganglion - Skeletal Muscle Coculture of Embryonic Rat. I. The Morphological Correlates of the Spinal Reflex Arc.

The cytoarchitecture of a spinal cord - dorsal root ganglion - skeletal muscle tissue coculture system was investigated at the level of the light microscope using a number of different staining techniques. In these cultures central synapses between dorsal root ganglion (DRG) cells and interneurons in the ventral spinal cord and between DRG cells and motoneurons were visualized by parvalbumin immunostaining and by intracellular horseradish peroxidase (HRP) filling of DRG cells. Skeletal muscle fibres regenerated in vitro first into multinucleated myotubes, and around day 8 in vitro into well differentiated muscle fibres with regular cross-striation. At the same time newly formed motor endplates could be visualized using acetylcholinesterase staining. The axons of motoneurons could be traced retrogradely by local application of HRP to the regenerated muscle fibres. The motor axons sometimes gave off collaterals reminiscent of Renshaw collaterals at about 300 microm from the axon hillock. Intracellular filling to motoneurons with HRP revealed that only a minority of the motoneurons within a culture had reached their appropriate target. Comparing the dendrograms of the motoneurons which had innervated muscles to those which had not suggested that motoneurons innervating muscle tissue had more complex dendritic trees and larger somata than those which did not innervate muscle tissue. Peripheral neurites of parvalbumin-immunoreactive DRG cells coiling around regenerated muscle fibres could be demonstrated in these cultures. These probably correspond to that part of the sensory muscle spindle apparatus which developed in vivo. However, only a few of the several hundred DRG cells found in every culture were parvalbumin-immunoreactive, suggesting that the actual number of Ia and II afferents within the population of DRG cells in culture is very small. This study demonstrates that all the neural elements necessary for the segmental spinal reflexes develop and can be maintained for several weeks in vitro.

An Organotypic Spinal Cord - Dorsal Root Ganglion - Skeletal Muscle Coculture of Embryonic Rat. II. Functional Evidence for the Formation of Spinal Reflex Arcs In Vitro.

Electrical properties of motoneurons, muscle fibres and dorsal root ganglion (DRG) cells were studied in an organotypic coculture of embryonic rat spinal cord, dorsal root ganglia and skeletal muscle. The motoneurons were identified by their morphology and position in culture. Their size and input conductance were significantly larger than those of spinal interneurons. Intracellular current injection evoked action potentials in all motoneurons, but only evoked stable repetitive firing patterns in some. Excitability was correlated to somatic size and the rate of spontaneous excitatory input. It is suggested that the somatic growth and the increase in excitability is regulated by the excitatory afferents. The motoneurons showed spontaneous excitatory and inhibitory postsynaptic potentials and action potentials which disappeared with the application of various agents known to inhibit excitability or excitatory synaptic transmission. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs respectively) were distinguished by their shape, reversal potential and pharmacology. IPSPs could be depolarizing or hyperpolarizing in different cells. A higher percentage of cells with hyperpolarizing IPSPs was found in older cultures and in the presence of skeletal muscle, suggesting a reversal of the polarity of IPSPs with development. The spontaneous muscle contractions observed in the cultures could be due either to innervation, spontaneous oscillations of the membrane potential, or electrical coupling between neighbouring fibres. A small percentage of DRG cells showed spontaneous action potentials, all of which were found in cultures with spontaneous muscle contractions. The electrical stimulation of DRG afferents evoked mono- and polysynaptic EPSPs in motoneurons, endplate potentials and muscle contractions. The stimulation of the ventral horns evoked endplate potentials and muscle contractions via mono- or polysynaptic pathways. Together these results indicate that appropriate and functional contacts were established in the culture between myotubes and DRG cells, between DRG cells and motoneurons, and between motoneurons and muscle fibres.